Ectromelia virus (ECTV), the cause of mousepox, is the prototypical representative of a large class of pathogens that invade the host via lymphohematogenous spread and an outstanding model for the study of natural virus:host interplay. ECTV poses a formidable challenge to the host and numerous arms of the host defense system are required to bring it under control. This includes CD4+ T cells (TCD4+) that, through recognition of foreign peptides (epitopes) in combination with Major Histocompatility Complex class II molecules (MHCII), guide and potentiate adaptive responses and, in some cases, carry out their own effector functions. Much of what we understand about TCD4+ activation, effector function and memory has been deduced through the use of nominal protein antigens with limited relevance to bona fide pathogens. In fact, preliminary and established data indicate that TCD4+ activation is a complex proposition with the possibility of peptide production via an array of alternative pathways, all of which are quite distinct from the classical textbook versions of antigen processing, and which we hypothesize to be major determinants of TCD4+ expansion and functionality. This notion will be tested in our next steps towards gaining a comprehensive and fundamental understanding of the TCD4+ response in a natural virus-host relationship, our long-term goal. To these ends we will: 1) markedly expand the number of known peptide sequences that induce TCD4+ responses to ECTV in C57Bl/6 (B6) mice and determine the processing pathways by which they are generated, 2) identify the antigen presenting cells that present particular ECTV epitopes, and 3) determine impact of processing pathway on TCD4+ expansion and functionality. The proposed experiments could revise fundamental principles of adaptive immunity, thereby leading to innovations in rational vaccine design.